Extraction of total RNA from FFPE tissue
PROTOCOL FOR:
Methods comparison for high resolution transcriptional analysis of archival
material on Affymetrix Plus 2.0 and Exon 1.0 microarrays
Kim M. Linton1,5*, Yvonne Hey2*, Sian Dibben2, Crispin J. Miller3, Anthony J. Freemont4,
John A. Radford1,5, Stuart D. Pepper2
1
Cancer Research UK Department of Medical Oncology, The Christie NHS Foundation Trust,
Manchester, UK, 2Molecular Biology Core Facility, Cancer Research UK, Paterson Institute
for Cancer Research, The University of Manchester, Manchester, UK, 3Applied Computational
Biology and Bioinformatics Group, Cancer Research UK, Paterson Institute for Cancer
Research, The University of Manchester, Manchester, UK, 4School of Clinical & Laboratory
Sciences, The University of Manchester, Manchester, UK, and 5School of Cancer and Imaging
Sciences, The University of Manchester, Manchester, UK
LEGEND
ATTENTION
* HINT
REST
This is a modification of the Optimum FFPE RNA Isolation Kit protocol (Ambion,
Huntingdon, UK). This kit has now been discontinued.
REAGENTS
 Xylene (AnalaR) (BDH Laboratory Supplies, Poole, Dorset, UK)
 Ethyl alcohol/Ethanol (absolute) (Sigma-Aldrich, Poole, Dorset, UK)
 Proteinase K solutiona
 Proteinase K digestion buffer, warmed to room temperaturea
 RNA extraction buffera
 Wash solution 1a
 Wash solution 2a
 Wash solution 3a
 Elution solutiona
a
Reagents supplied with Optimum FFPE RNA Isolation Kit (Ambion, Huntingdon, UK)
PROCEDURE
1. Add 1 mL xylene to each tube and vortex vigorously for 10 s to deparaffinize.
2. Incubate at 20°C for 10–20 min.
3. Spin at maximum speed for 3 min to pellet tissue and remove xylene by pipetting
4. Repeat deparaffinization steps 1–3.
5. Add 1 mL 100% ethanol to wash sections and vortex vigorously for 10 s.
6. Spin at maximum speed for 2 min to pellet tissue and remove ethanol by pipetting.
7. Repeat with one wash in 90% ethanol followed by one wash in 70% ethanol.
8. Re-spin at 13,400 – 16,500× g and remove residual ethanol by pipetting with a fine
bore tip; repeat this step until no visible ethanol remains.
9. Open caps and allow sections to dry completely (if necessary, place tubes in vented
hood to aid drying).
10. Prepare a master mix at room temperature. For the first RNA extraction, use the
following amounts per reaction:
10 μL Proteinase K solution
100 μL Proteinase K digestion buffer, warmed to room temperature
115 μL Total
*For samples where RNA extraction has previously been attempted and where a large
amount of undigested tissue was present at the end of proteinase K incubation (step 13),
use the following amounts per reaction to prepare a master mix
20 μL Proteinase K solution
130 μL Proteinase K digestion buffer, warmed to room temperature
150 μL Total
11. Add 115/150 μL of master mix to each sample and vortex vigorously for 10–20 s to
disperse the tissue.
12. Incubate at 37°C for 2–6 hours and flick tube several times to aid mechanical
disruption of tissues.
13. Incubate at 55°C for a further 1–2 hours if there is residual undissolved tissue
14. Spin at 13,400 – 16,500× g for 1 min to pellet insoluble tissue and transfer clear
preparation to a new tube.
15. Add 200 μL RNA extraction buffer (warmed to room temperature) to each sample and
vortex vigorously for 10 s; spin briefly to collect contents at the bottom of the tube.
16. Add 160 μL 100% ethanol to each sample and vortex vigorously for 10 s; spin briefly to
collect contents at the bottom of the tube.
17. Apply half the sample (235 μL) to the filter cartridge in a collection tube (filter
cartridge assembly) and spin at 13,400 – 16,500× g for 1 min.
18. Repeat with the remaining sample.
19. Remove the filter cartridge, discard the filtrate and replace the filter cartridge in the
collection tube.
20. Apply 180 μL of wash solution 1 to the filter cartridge assembly, cut lid off the
collection tube, and spin at 13,400 – 16,500× g for 1 min or until all the liquid has
passed through the filter.
21. Apply 180 μL of wash solution 2/3 to the filter cartridge assembly, and spin at
maximum speed for 1 min or until all the liquid has passed through the filter.
22. Repeat with a second 180 μL of wash solution 2/3.
23. Remove the filter cartridge, discard the filtrate and replace the filter cartridge into the
collection tube.
24. Spin the filter cartridge assembly at maximum speed for 5 min, place the filter cartridge
into an elution tube and discard the collection tube.
25. Pipette 20 μL of Elution Solution (heated to 70°C) onto the centre of the filter, wait 1
min and spin at maximum speed for 2 min to elute.
26. Re-apply the 20 μL eluate to the filter, then turn the columns around 180° and spin for
a further 2 min at maximum speed to elute.
27. Proceed to ‘DNase 1 treatment of total RNA’ or store at -80°C until DNase treatment
can be carried out.
EQUIPMENT
Centrifuge
Glassware
Pipet
Filter cartridge assembly
Filter cartridge
Vortexer
Fine-bore pipet tips